Tandem fluorescent protein constructs

ABSTRACT

This invention provides tandem fluorescent protein construct including a donor fluorescent protein moiety, an acceptor fluorescent protein moiety and a linker moiety that couples the donor and acceptor moieties. The donor and acceptor moieties exhibit fluorescence resonance energy transfer which is eliminated upon cleavage. The constructs are useful in enzymatic assays.

This application is a continuation-in-part of U.S. Ser. No. 08/594,575,filed Jan. 31, 1996.

BACKGROUND OF THE INVENTION

Proteases play essential roles in many disease processes such asAlzheimer's, hypertension, inflammation, apoptosis, and AIDS. Compoundsthat block or enhance their activity have potential as therapeuticagents. Because the normal substrates of peptidases are linear peptidesand because established procedures exist for making non-peptidicanalogs, compounds that effect the activity of proteases are naturalsubjects of combinatorial chemistry. Screening compounds produced bycombinatorial chemistry requires convenient enzymatic assays.

The most convenient existing assays for proteases are based onfluorescence resonance energy transfer from a donor fluorophore to aquencher placed at opposite ends of a short peptide chain containing thepotential cleavage site. Knight C G, "Fluorimetric assays of proteolyticenzymes," Methods in Enzymol. (1995) 248:18-34. Proteolysis separatesthe fluorophore and quencher, resulting in increased intensity in theemission of the donor fluorophore. Existing protease assays use shortpeptide substrates incorporating unnatural chromophoric amino acids,assembled by solid phase peptide synthesis. However, solid phasesynthesis poses certain problems of effort and expense.

It is useful to perform enzymatic assays in vivo, in order to moreclosely mimic conditions in which intracellular proteases act.Conventional artificial substrates prepared by solid-phase synthesiswould require microinjection into individual cells, which is impracticalas a high-throughput screen. Also, short unfolded peptides are generallyrapidly degraded by nonspecific mechanisms inside cells.

The Edans fluorophore is the current mainstay of existing fluorometricassays. Fluorophores with greater extinction coefficients and quantumyields are desirable. The Edans fluorophore often is coupled with anon-fluorescent quencher such as Dabcyl. However, assays performed withsuch agents rely on the absolute measurement of fluorescence from thedonor. This amount is contaminated by other factors including turbidityor background absorbances of the sample, fluctuations in the excitationintensity, and variations in the absolute amount of substrate.

SUMMARY OF THE INVENTION

This invention provides tandem fluorescent protein constructs andmethods for using them in enzymatic assays both in vitro and in vivo.Tandem fluorescent protein constructs comprise a donor fluorescentprotein moiety, an acceptor fluorescent protein moiety and a linkermoiety that couples the donor and acceptor moieties, wherein the donorand acceptor moieties exhibit fluorescence resonance energy transferwhen the donor moiety is excited. The fluorescent protein moieties canbe Aequorea-related fluorescent protein moieties, such as greenfluorescent protein and blue fluorescent protein. In one aspect, thelinker moiety comprises a cleavage recognition site for an enzyme, andis, preferably, a peptide of between 5 and 50 amino acids. In oneembodiment, the construct is a fusion protein in which the donor moiety,the peptide moiety and the acceptor moiety are part of a singlepolypeptide.

This invention also provides recombinant nucleic acids coding forexpression of tandem fluorescent protein constructs in which a donorfluorescent protein moiety, an acceptor fluorescent protein moiety and apeptide linker moiety are encoded in a single polypeptide. The inventionalso provides expression vectors comprising expression control sequencesoperatively linked to a recombinant nucleic acid coding for theexpression of a tandem fluorescent protein construct, as well as hostcells transfected with those expression vectors.

The tandem constructs of this invention are useful in assays fordetermining whether a sample contains an enzyme. The methods involvecontacting the sample with a tandem fluorescent protein construct. Thedonor moiety is excited. Then the degree of fluorescence resonanceenergy transfer in the sample is determined. A degree of fluorescenceresonance energy transfer that is lower than an expected amountindicates the presence of an enzyme. The degree of fluorescenceresonance energy transfer in the sample can be determined as a functionof the amount of fluorescence from the donor moiety, the amount offluorescence from the acceptor donor moiety, the ratio of the amount offluorescence from the donor moiety to the amount of fluorescence fromthe acceptor moiety or the excitation state lifetime of the donormoiety.

The assay also is useful for determining the amount of enzyme in asample by determining the degree of fluorescence resonance energytransfer at a first and second time after contact between the enzyme andthe tandem construct, and determining the difference in the degree offluorescence resonance energy transfer. The difference in the degree offluorescence resonance energy transfer reflects the amount of enzyme inthe sample.

The invention also provides methods for determining the amount ofactivity of an enzyme in a cell. The methods involve providing a cellthat expresses a tandem fluorescent protein construct, for example bytransfecting the cell with an appropriate expression vector. The cell isexposed to light in order to excite the donor moiety. Then the degree offluorescence resonance energy transfer in the cell is determined. Thedegree of fluorescence resonance energy transfer reflects to the amountof enzyme activity in the cell.

Similarly, the invention provides methods of determining the amount ofactivity of an enzyme in a sample from an organism. The methods involveproviding a sample from an organism having a cell that expresses atandem fluorescent protein construct. The donor moiety in the sample isexcited. Then the degree of fluorescence resonance energy transfer inthe sample is determined. The degree of fluorescence resonance energytransfer reflects the amount of enzyme activity in the cell.

The assay methods also can be used to determine whether a compoundalters the activity of an enzyme, i.e., screening assays. The methodsinvolve contacting a sample containing an amount of the enzyme with thecompound and with a tandem fluorescent protein construct; exciting thedonor moiety; determining the amount of enzyme activity in the sample asa function of the degree of fluorescence resonance energy transfer inthe sample; and comparing the amount of activity in the sample with astandard activity for the same amount of the enzyme. A differencebetween the amount of enzyme activity in the sample and the standardactivity indicates that the compound alters the activity of the enzyme.

Similar methods, are useful for determining whether a compound altersthe activity of an enzyme in a cell. The methods involve providing firstand second cells that express a tandem fluorescent protein construct;contacting the first cell with an amount of the compound; contacting thesecond cell with a different amount of the compound; exciting the donormoiety in the first and second cell; determining the degree offluorescence resonance energy transfer in the first and second cells;and comparing the degree of fluorescence resonance energy transfer inthe first and second cells. A difference in the degree of fluorescenceresonance energy transfer indicates that the compound alters theactivity of the enzyme.

Assays of the invention are also useful for determining andcharacterizing substrate cleavage sequences of proteases or foridentifying proteases, such as orphan proteases. In one embodiment themethod involves the replacement of a defined linker moiety amino acidsequence with one that contains a randomized selection of amino acids. Alibrary of fluorescent protein moieties each linked by a randomizedlinker moiety can be generated using recombinant engineering techniquesor synthetic chemistry techniques. Screening the members of the librarycan be accomplished by measuring a signal related to cleavage, such asfluorescence energy transfer, after contacting the cleavage enzyme witheach of the library members of the tandem fluorescent protein construct.A degree of fluorescence resonance energy transfer that is lower than anexpected amount indicates the presence of a linker sequence that can becleaved by the enzyme. The degree of fluorescence resonance energytransfer in the sample can be determined as a function of the amount offluorescence from the donor moiety, the amount of fluorescence from theacceptor donor moiety, or the ratio of the amount of fluorescence fromthe donor moiety to the amount of fluorescence from the acceptor moietyor the excitation state lifetime of the donor moiety.

Libraries of fluorescent proteins can be expressed in cells and used tocharacterize the recognition motif of proteases expressed within cells,where the enzyme is in its native context. This method provides theadditional advantage of assessing the specificity of any given linkersequence to cleavage by other enzymes other than the target enzyme. Themethods consist of the generation of a library of recombinant hostcells, each of which expresses a tandem fluorescent protein constructlinked through a randomized candidate linker substrate. Each cell isexpanded into a clonal population that is genetically homogeneous andthe degree of energy transfer is measured from each clonal population.Optionally, FRETS can be measured before and at least one specified timeafter a known change in intracellular protease activity. A change in thedegree of fluorescence resonance energy transfer demonstrates that thecell contains a tandem construct and linker sequence that can be cleavedby the enzyme activity in the cell. Such methods are particular suitedto Fluorescent Activated Cell Sorter (FACS) clonal selection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence (SEQ ID NO:1) and deduced aminoacid sequence (SEQ ID NO:2) of a wild-type Aequorea green fluorescentprotein.

FIG. 2 depicts a tandem construct of the invention involved in FRET.

FIG. 3 depicts fluorescence emission spectra of a composition containinga tandem S65C--linker--P4-3 fluorescent protein construct excited at 368nm after exposure to trypsin for 0, 2, 5, 10 and 47 minutes.

FIG. 4 depicts fluorescence emission spectra intensity of a compositioncontaining a tandem S65C--linker--P4-3 fluorescent protein constructexcited at 368 nm after exposure to calpain for 0, 2, 6 and 15 minutes.

FIG. 5 depicts fluorescence emission spectra of a composition containinga tandem S65C--linker--P4 fluorescent protein construct excited at 368nm after exposure to enterokinase for 0, 2, 20 and 144 minutes.

FIG. 6 depicts fluorescence emission spectra of a composition containinga tandem S65T--linker--W7 fluorescent protein construct excited at 432nm before and after exposure to trypsin.

FIG. 7 depicts fluorescence emission spectra of a composition containinga tandem P4-3--linker--W7 fluorescent protein construct excited at 368nm before and after exposure to trypsin.

FIG. 8 depicts fluorescence emission spectra of a composition containinga tandem W1B--linker--10c fluorescent protein construct excited at 433nm before and after exposure to trypsin.

FIG. 9 depicts the time course of fluorescent ratio changes uponcleavage of a composition containing the tandem W1B--linker--10cfluorescent protein construct measured at different proteinconcentrations after exposure to trypsin measured in a fluorescent 96well plate reader.

FIG. 10 depicts a method of generating fluorescent tandem constructsseparated by a randomized linker region for use in identifying cleavagespecificities or orphan proteases.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the laboratory procedures in cell culture, moleculargenetics, and nucleic acid chemistry and hybridization described beloware those well known and commonly employed in the art. Standardtechniques are used for recombinant nucleic acid methods, polynucleotidesynthesis, and microbial culture and transformation (e.g.,electroporation, lipofection). Generally, enzymatic reactions andpurification steps are performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. Molecular Cloning: ALaboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference)which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, organicsynthetic chemistry, and pharmaceutical formulation described below arethose well known and commonly employed in the art. Standard techniquesare used for chemical syntheses, chemical analyses, pharmaceuticalformulation and delivery, and treatment of patients. As employedthroughout the disclosure, the following terms, unless otherwiseindicated, shall be understood to have the following meanings:

"Moiety" refers to the radical of a molecule that is attached to anothermoiety. Thus, a "fluorescent protein moiety" is the radical of afluorescent protein coupled to the linker moiety. By the same token, theterm "linker moiety" refers to the radical of a molecular linker that iscoupled to both the donor and acceptor protein moieties.

"Fluorescent protein" refers to any protein capable of fluorescence whenexcited with appropriate electromagnetic radiation. This includesfluorescent proteins whose amino acid sequences are either natural orengineered.

"Peptide" refers to a polymer in which the monomers are amino acids andare joined together through amide bonds, alternatively referred to as apolypeptide. When the amino acids are a-amino acids, either theL-optical isomer or the D-optical isomer may be used. Additionally,unnatural amino acids, for example, b-alanine, phenylglycine andhomoarginine are also meant to be included. Commonly encountered aminoacids which are not gene-encoded may also be used in the presentinvention. All of the amino acids used in the present invention may beeither the D- or L-isomer. The L-isomers are preferred. In addition,other peptidomimetics are also useful in the linker moieties of thepresent invention. For a general review see Spatola, A. F., in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, B. Weinstein,eds., Marcel Dekker, New York, p. 267 (1983).

"Naturally-occurring" as used herein, as applied to an object, refers tothe fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

"Operably linked" refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A control sequence "operably linked" to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequences.

"Control sequence" refers to polynucleotide sequences which arenecessary to effect the expression of coding and non-coding sequences towhich they are ligated. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term"control sequences" is intended to include, at a minimum, componentswhose presence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

"Polynucleotide" refers to a polymeric form of nucleotides of at least10 bases in length, either ribonucleotides or deoxynucleotides or amodified form of either type of nucleotide. The term includes single anddouble stranded forms of DNA.

"Modulation" refers to the capacity to either enhance or inhibit afunctional property of biological activity or process (e.g., enzymeactivity or receptor binding); such enhancement or inhibition may becontingent on the occurrence of a specific event, such as activation ofa signal transduction pathway, and/or may be manifest only in particularcell types.

The term "modulator" refers to a chemical compound (naturally occurringor non-naturally occurring), such as a biological macromolecule (e.g.nucleic acid, protein, non-peptide, or organic molecule), or an extractmade from biological materials such as bacteria, plants, fungi, oranimal (particularly mammalian) cells or tissues. Modulators areevaluated for potential activity as inhibitors or activators (directlyor indirectly) of a biological process or processes (e.g., agonist,partial antagonist, partial agonist, antagonist, antineoplastic agents,cytotoxic agents, inhibitors of neoplastic transformation or cellproliferation, cell proliferation-promoting agents, and the like) byinclusion in screening assays described herein. The activities (oractivity) of a modulator may be known, unknown or partial known. Suchmodulators can be screened using the methods described herein.

The term "test compound" refers to a compound to be tested by one ormore screening method(s) of the invention as a putative modulator.Usually, various predetermined concentrations are used for screeningsuch as 0.01 uM, 0.1 uM, 1.0 uM, and 10.0 uM. Test compound controls caninclude the measurement of a signal in the absence of the test compoundor comparison to a compound known to modulate the target.

INTRODUCTION

It has been discovered that fluorescent proteins having the properemission and excitation spectra that are brought into physically closeproximity with one another can exhibit fluorescence resonance energytransfer ("FRET"). This invention takes advantage of that discovery toprovide tandem fluorescent protein constructs in which two fluorescentprotein moieties capable of exhibiting FRET are coupled through a linkerto form a tandem construct. The protein moieties are chosen such thatthe excitation spectrum of one of the moieties (the acceptor moiety)overlaps with the emission spectrum of the excited protein moiety (thedonor moiety). The donor moiety is excited by light of appropriateintensity within the donor's excitation spectrum. The donor then emitsthe absorbed energy as fluorescent light. The fluorescent energy itproduces is quenched by the acceptor fluorescent protein moiety. FRETcan be manifested as a reduction in the intensity of the fluorescentsignal from the donor, reduction in the lifetime of its excited state,and re-emission of fluorescent light at the longer wavelengths (lowerenergies) characteristic of the acceptor. When the linker that connectsthe donor and acceptor moieties is cleaved, the fluorescent proteinsphysically separate, and FRET is diminished or eliminated. This has alsobeen described in U.S. patent application Ser. No. 08/594,575, filedJan. 31, 1996, which is herein incorporated by reference.

One can take advantage of the FRET exhibited by the tandem fluorescentprotein constructs of the invention in performing enzymatic assays. Anembodiment of this process is depicted in FIG. 2. A recombinant nucleicacid encodes a single polypeptide including a poly-histidinyl tag, ablue fluorescent protein donor moiety, a peptide linker moietycomprising a protease recognition site and a green fluorescent proteinacceptor moiety. The nucleic acid can be expressed into a tandemfluorescent protein construct of the invention. In this example, atandem construct contains a blue fluorescent protein (such as P4-3,TABLE I) as the donor moiety and a green fluorescent protein (such asS65C, TABLE I) as the acceptor moiety.

The construct is exposed to light at, for example, 368 nm, a wavelengththat is near the excitation maximum of P4-3. This wavelength excitesS65C only minimally. Upon excitation, some portion of the energyabsorbed by the blue fluorescent protein moiety is transferred to theacceptor moiety through FRET. As a result of this quenching, the bluefluorescent light emitted by the blue fluorescent protein is less brightthan would be expected if the blue fluorescent protein existed inisolation. The acceptor moiety (S65C) may re-emit the energy at longerwavelength, in this case, green fluorescent light.

After cleavage of the linker moiety by an enzyme, the blue and greenfluorescent proteins physically separate and FRET is lost. Over time, asincreasing amounts of the tandem construct are cleaved, the intensity ofvisible blue fluorescent light emitted by the blue fluorescent proteinincreases, while the intensity of visible green light emitted by thegreen fluorescent protein as a result of FRET, decreases.

The tandem fluorescent protein constructs of this invention are usefulas substrates to study agents or conditions that cleave the linker. Inparticular, this invention contemplates tandem constructs in which thelinker is a peptide moiety containing an amino acid sequence that is acleavage site for a protease of interest. The amount of the protease ina sample is determined by contacting the sample with a tandemfluorescent protein construct and measuring changes in fluorescence ofthe donor moiety, the acceptor moiety or the relative fluorescence ofboth. In one embodiment, the tandem construct is a recombinant fusionprotein produced by expression of a nucleic acid that encodes a singlepolypeptide containing the donor moiety, the peptide linker moiety andthe acceptor moiety. Fusion proteins can be used for, among otherthings, monitoring the activity of a protease inside the cell thatexpresses the recombinant tandem construct. The distance betweenfluorescent proteins in the construct can be regulated based on thelength of the linking moiety. Therefore, tandem constructs of thisinvention whose linker moieties do not include cleavage sites also areuseful as agents for studying FRET between fluorescent proteins.

Advantages of tandem fluorescent protein constructs include the greaterextinction coefficient and quantum yield of many of these proteinscompared with those of the Edans fluorophore. Also, the acceptor in atandem construct is, itself, a fluorophore rather than a non-fluorescentquencher like Dabcyl. Thus, the enzyme's substrate (i.e., the tandemconstruct) and products (i.e., the moieties after cleavage) are bothfluorescent but with different fluorescent characteristics.

In particular, the substrate and cleavage products exhibit differentratios between the amount of light emitted by the donor and acceptormoieties. Therefore, the ratio between the two fluorescences measuresthe degree of conversion of substrate to products, independent of theabsolute amount of either, the optical thickness of the sample, thebrightness of the excitation lamp, the sensitivity of the detector, etc.Furthermore, the Aequorea-related fluorescent protein moieties tend tobe protease resistant. Therefore, they are likely to survive asfluorescent moieties even after the linker moiety is cleaved.

II. TANDEM FLUORESCENT PROTEIN CONSTRUCTS

The tandem fluorescent protein constructs of the invention usuallycomprise three elements: a donor fluorescent protein moiety, an acceptorfluorescent protein moiety and a linker moiety that couples the donorand acceptor moieties. The donor fluorescent protein moiety is capableof absorbing a photon and transferring energy to another fluorescentmoiety. The acceptor fluorescent protein moiety is capable of absorbingenergy and emitting a photon. The linker moiety connects the donorfluorescent protein moiety to the acceptor fluorescent protein moiety.In many instances the linker moiety will covalently connect the donorfluorescent protein moiety and the acceptor fluorescent protein moiety.It is desirable, as described in greater detail herein, to select adonor fluorescent protein moiety with an emission spectrum that overlapswith the excitation spectrum of an acceptor fluorescent protein moiety.In some embodiments of the invention the overlap in emission andexcitation spectra will facilitate FRET, Such an overlap is notnecessary, however, if intrinsic fluorescence is measured instead ofFRET. Any fluorescent protein may be used in the invention, includingproteins that have fluoresce due intramolecular rearrangements or theaddition of cofactors that promote fluorescence.

For example, green fluorescent proteins ("GFPs") of cnidarians, whichact as their energy-transfer acceptors in bioluminescence, can be usedin the invention. A green fluorescent protein, as used herein, is aprotein that fluoresces green light, and a blue fluorescent protein is aprotein that fluoresces blue light. GFPs have been isolated from thePacific Northwest jellyfish, Aequorea victoria, the sea pansy, Renillareniformis, and Phialidium gregarium. W. W. Ward et al., Photochem.Photobiol., 35:803-808 (1982); L. D. Levine et al., Comp. Biochem.Physiol., 72B:77-85 (1982).

A variety of Aequorea-related GFPs having useful excitation and emissionspectra have been engineered by modifying the amino acid sequence of anaturally occurring GFP from Aequorea victoria. (D. C. Prasher et al.,Gene, 111:229-233 (1992); R. Heim et al., Proc. Natl. Acad. Sci., USA,91:12501-04 (1994); U.S. patent application Ser. No. 08/337,915, filedNov. 10, 1994; International application PCT/US95/14692, filed Nov. 10,1995; U.S. patent application Ser. No. 08/706,408, filed Aug. 30,1996.The green fluorescent protein (GFP) of the jellyfish Aequoreavictoria is a remarkable protein with strong visible absorbance andfluorescence from a p-hdroxybenzlideneimidazolone chromophore, which isgenerated by cyclization and oxidation of the protein's own Ser-Tyr-Glysequence at positions 65 to 67. A cDNA sequence for one isotype of GFPhas been reported [Prasher, D. C. et al., Gene 111, 229-233 (1992)]. Thefinding that the expressed protein becomes fluorescent in cells from awide variety of organisms [Chalfie, M. et al., Science 263, 802-805(1994)] makes GFP a powerful new tool in molecular and cell biology andindicates that the oxidative cyclization must be either spontaneous ordependent only on ubiquitous enzymes and reactants. As used herein, afluorescent protein is an Aequorea-related fluorescent protein if anycontiguous sequence of 150 amino acids of the fluorescent protein has atleast 85% sequence identity with an amino acid sequence, eithercontiguous or non-contiguous, from the wild type Aequorea greenfluorescent protein of SEQ ID NO:2. More preferably, a fluorescentprotein is an Aequorea-related fluorescent protein if any contiguoussequence of 200 amino acids of the fluorescent protein has at least 95%sequence identity with an amino acid sequence, either contiguous ornon-contiguous, from the wild type Aequorea green fluorescent protein ofSEQ ID NO:2. Similarly, the fluorescent protein may be related toRenilla or Phialidium wild-type fluorescent proteins using the samestandards.

Aequorea-related fluorescent proteins include, for example, wild-type(native) Aequorea victoria GFP, whose nucleotide (SEQ ID NO:1) anddeduced amino acid (SEQ ID NO:2) sequences are presented in FIG. 1; andthose Aequorea-related engineered versions described in TABLE I. Severalof these, i.e., P4, P4-3, W7 and W2 fluoresce at a distinctly shorterwavelength than wild type.

                                      TABLE I                                     __________________________________________________________________________                             Extinct.                                                                                 Excitation Emission max Coefficient                                        Quantum                                        Clone Mutation(s) max (nm) (nm) (M                                                                           .sup.-1 cm.sup.-1) yield                     __________________________________________________________________________    Wild type                                                                          None  393 (475)                                                                           508     21,000 (7,150)                                                                        0.77                                           P4 Y66H 383 447 13,500 0.21                                                   P4-3 Y66H 381 445 14,000 0.38                                                  Y145F                                                                        W7 Y66W 433 (453) 475 (501) 18,000 0.67                                        N146L   (17,100)                                                              M153T                                                                         V163A                                                                         N212K                                                                        W2 Y66W 432 (453) 480 10,000 (9,600) 0.72                                      I123V                                                                         Y145H                                                                         H148R                                                                         M153T                                                                         V163A                                                                         N212K                                                                        S65T S65T 489 511 39,200 0.68                                                 P4-I S65T 504 (396) 514 14,500 (8,600) 0.53                                    M153A                                                                         K238E                                                                        S65A S65A 471 504                                                             S65C S65C 479 507                                                             S65L S65L 484 510                                                             Y66F Y66F 360 442                                                             Y66W Y66W 458 480                                                             10c S65G 513 527                                                               V68L                                                                          V72A                                                                          T203Y                                                                        W1B F64L 432 (453) 476 (503)                                                   S65T                                                                          Y66W                                                                          N146I                                                                         M153T                                                                         V163A                                                                         N212K                                                                        Emerald S65T 487 508                                                           S72A                                                                          N149K                                                                         M153T                                                                         I167T                                                                        Sapphire S72A 395 511                                                          Y145F                                                                         T203I                                                                      __________________________________________________________________________

This invention contemplates the use of other fluorescent proteins intandem constructs. The cloning and expression of yellow fluorescentprotein from Vibrio fischeri strain Y-1 has been described by T. O.Baldwin et al., Biochemistry. (1990) 29:5509-15. This protein requiresflavins as fluorescent co-factors. The cloning of Peridinin-chlorophylla binding protein from the dinoflagellate Symbiodinium sp. was describedby B. J. Morris et al., Plant Molecular Biology, (1994) 24:673:77. Oneuseful aspect of this protein is that it fluoresces in red. The cloningof phycobiliproteins from marine cyanobacteria such as Synechococcus,e.g., phycoerythrin and phycocyanin, is described in S. M. Wilbanks etal., J. Biol. Chem. (1993) 268:1226-35. These proteins requirephycobilins as fluorescent co-factors, whose insertion into the proteinsinvolves auxiliary enzymes. The proteins fluoresce at yellow to redwavelengths.

For FRET, the donor fluorescent protein moiety and the acceptorfluorescent protein moiety are selected so that the donor and acceptormoieties exhibit fluorescence resonance energy transfer when the donormoiety is excited. One factor to be considered in choosing thefluorescent protein moiety pair is the efficiency of fluorescenceresonance energy transfer between them. Preferably, the efficiency ofFRET between the donor and acceptor moieties is at least 10%, morepreferably at least 50% and even more preferably at least 80%. Theefficiency of FRET can easily be empirically tested using the methodsdescribed herein and known in the art, particularly, using theconditions set forth in the Examples.

The efficiency of FRET is dependent on the separation distance and theorientation of the donor and acceptor moieties, as described by theForster equation, the fluorescent quantum yield of the donor moiety andthe energetic overlap with the acceptor moiety. Forster derived therelationship:

    E=(F.sup.0 -F)/F.sup.0 =R.sub.0.sup.6 /(R.sup.6 +R.sub.0.sup.6)

where E is the efficiency of FRET, F and F⁰ are the fluorescenceintensities of the donor in the presence and absence of the acceptor,respectively, and R is the distance between the donor and the acceptor.R₀, the distance at which the energy transfer efficiency is 50%, isgiven (in Å) by

    R.sub.0 =9.79×10.sup.3 (K.sup.2 QJn.sup.-4).sup.1/6

where K² is an orientation factor having an average value close to 0.67for freely mobile donors and acceptors, Q is the quantum yield of theunquenched fluorescent donor, n is the refractive index of theintervening medium, and J is the overlap integral, which expresses inquantitative terms the degree of spectral overlap,

    J=∫.sup.∞.sub.0 ε.sub.λ F.sub.λ λ.sup.4 dλ/∫.sup.∞.sub.0 F.sub.λ dλ

where ε.sub.λ is the molar absorptivity of the acceptor in M⁻¹ cm⁻¹ andF.sub.λ is the donor fluorescence at wavelength l measured in cm.Forster, T. (1948) Ann. Physik 2:55-75. Tables of spectral overlapintegrals are readily available to those working in the field (forexample, Berlman, I. B. Energy transfer parameters of aromaticcompounds, Academic Press, New York and London (1973)).

The characteristic distance R₀ at which FRET is 50% efficient depends onthe quantum yield of the donor i.e., the shorter-wavelength fluorophore,the extinction coefficient of the acceptor, i.e., the longer-wavelengthfluorophore, and the overlap between the donor's emission spectrum andthe acceptor's excitation spectrum. Calculated values of R₀ for P4-3 toS65T and S65C are both 4.03 nm because the slightly higher extinctioncoefficient of S65T compensates for its slightly longer emissionwavelength. R. Heim et al., "Improved green fluorescence," Nature (1995)373:663-664.

The efficiency of FRET between the two fluorescent proteins can also beadjusted by changing ability of the two fluorescent proteins to dimerizeor closely associate. If the two fluorescent proteins are known ordetermined to closely associate, an increase or decrease in dimerizationcan be promoted by adjusting the length of the linker moiety between thetwo fluorescent proteins. Such dimerization can change Λ², R, J, and Qand dimerization changes directly affect the fluorescence spectracompared to undimerized protein. Consequently, for FRET aspects of theinvention, the change in intrinsic fluorescence can be used to adjustthe amount of FRET between the donor and the acceptor, as well asdimerization induced changes in FRET distances. Such dimerizationinduced changes in FRET distance can be optimized for maximal changes inFRET upon cleavage of a linker moiety by empirically determining thelength of the linker moiety that produces the best FRET. Usually, suchlinkers will be comparable to a length of 14 to 30 amino acids.

The ability of two fluorescent proteins to dimerize could be increasedby selecting amino acid positions that interact in the dimer and makingchanges of the amino acids at such positions that increase thehydrophobic or ionic interactions, or decrease the steric repulsions.Conversely, ability of two fluorescent proteins to dimerize could bedecreased by selecting amino acid positions that interact in the dimerand making changes in the amino acids at such positions that decreasethe hydrophobic or ionic interaction, or increase the steric repulsions.Thus, intramolecular interactions responsible for the association offluorescent protein moieties in a tandem fluorescent protein orintermolecular interactions between two fluorescent proteins in freesolution can be enhanced or attenuated.

For example, Aequorea derived fluorescent proteins and related proteins,especially at high concentrations of free protein, exist as dimers. Thedimerization domain can be identified in the wild type protein using thecrystal structure. Yang, F., et al The Molecular structure of GreenFluorescent Protein. Nature. Biotech. (1996) 14 1246-1251. In the caseof wildtype GFP, the hydrophobic amino acids, Ala 206, Leu 221, and Phe223 interact during dimerization. The tendency of a tandem GFP (or twoGFPs in free solution)to non-covalently associate at these positionscould be increased by increasing the hydrophobicity of amino acids atpositions 206 or 221, thereby increasing the strength of hydrophobicinteractions between the two fluorescent proteins.

For example, replacement of Ala 206, or Leu 221 by any of the aminoacids, Val, Ile or Phe would increase their hydrophobicity, andpotentially strengthen the hydrophobic interaction between two GFPs.Alternatively, the amino acids could be changed to positively chargedamino acids in one fluorescent protein (for example lys or Arg) and tonegatively charged amino acids in the second fluorescent protein of theconstruct (for example Glu or Asp) thereby creating additionalelectrostatic interactions between two GFPs. Similarly the amino acidsTyr 39, Glu 142, Asn 144, Asn 146, Ser 147, Asn 149, Tyr 151, Arg 168,Asn 170, Glu 172, Tyr 200, Ser 202, Gln 204 and Ser 208 could be changedaccording to the methods described herein to enhance intramolecularinteractions between tandem fluorescent proteins or intermolecularinteractions between to GFPs in free solution.

The length of the linker moiety is chosen to optimize both FRET and thekinetics and specificity of enzymatic cleavage. The average distancebetween the donor and acceptor moieties should be between about 1 nm andabout 10 nm, preferably between about 1 nm and about 6 nm, and morepreferably between about 1 nm and about 4 nm. If the linker is tooshort, the protein moieties may sterically interfere with each other'sfolding or with the ability of the cleavage enzyme to attack the linker.In embodiments of the invention where dimerization is desired the linkerlength will typically be a length comparable the length of at least 12amino acids, preferably at least 18 amino acids and more preferably atleast 24 amino acids. Only in rare instances will the linker length belonger than the length of about 40 to 50 amino acids. However,embodiments of the invention comprise linker moieties having 150 to 200amino acids.

The effect of linker length on the ability of tandemly linkedfluorescent proteins to become fluorescent was determined for a modifiedGFP tandem protein, as shown in TABLE II. The modified GFP tandemprotein was expressed in bacteria and grown at 37(C.

                  TABLE II                                                        ______________________________________                                                     Fluorescence of 1.sup.st                                                                   Fluorescence of 2.sup.nd                              Linker Length in Fluorescent protein Fluorescent protein                      amino acids (Sapphire) (10C)                                                ______________________________________                                        12           6.8 × 10.sup.4                                                                       6.2 × 10.sup.4                                  14 8.9 × 10.sup.4 8.4 × 10.sup.4                                  16 1.1 × 10.sup.5 1.0 × 10.sup.5                                  18 1.3 × 10.sup.5 1.2 × 10.sup.5                                  20 1.5 × 10.sup.5 1.4 × 10.sup.5                                  22 2.9 × 10.sup.5 1.6 × 10.sup.5                                  24 1.1 × 10.sup.6 7.8 × 10.sup.4                                  25 2.0 × 10.sup.6 1.2 × 10.sup.6                                ______________________________________                                    

Tandem fluorescent proteins of the invention comprising the general formSapphire--linker--10C (10C is also known as Topaz) were expressed in thebacterial cells JM109 (DE3). The linker moiety was constructed withvariable numbers of amino acids to evaluate the influence of linker sizeon fluorescence development. The linker sequences of TABLE II aredescribed as SEQ ID NO.: 26 to 31, respectively. The composition of the25 amino acid linker is identical to that used in the tandem fluorescentprotein constructs in the Examples. After overnight growth at 37(C. thebacterial colonies were examined to determine their relativefluorescence by resuspension in PBS after normalization for the numberof bacteria present by measuring the optical density at 600 nm.

When the intramolecular dimerization of a tandem fluorescent proteinconstruct is preferred, the three dimensional structure and flexabilityof the linker should permit the fluorescent protein moieties toassociate. When the linker moiety contains a cleavage site, the lengthof the linker can be between about 5 and about 50 amino acids and morepreferably between about 12 and 30 amino acids. Longer linkers maycreate too many sites which are vulnerable to attack by enzymes otherthan the one being assayed.

To optimize the efficiency and detectability of FRET within the tandemfluorescent protein construct, several factors need to be balanced. Theemission spectrum of the donor moiety should overlap as much as possiblewith the excitation spectrum of the acceptor moiety to maximize theoverlap integral J. Also, the quantum yield of the donor moiety and theextinction coefficient of the acceptor should likewise be as high aspossible to maximize R₀. However, the excitation spectra of the donorand acceptor moieties should overlap as little as possible so that awavelength region can be found at which the donor can be excitedefficiently without directly exciting the acceptor. Fluorescence arisingfrom direct excitation of the acceptor is difficult to distinguish fromfluorescence arising from FRET. Similarly, the emission spectra of thedonor and acceptor moieties should overlap as little as possible so thatthe two emissions can be clearly distinguished. High fluorescencequantum yield of the acceptor moiety is desirable if the emission fromthe acceptor is to be measured either as the sole readout or as part ofan emission ratio. In a preferred embodiment, the donor moiety isexcited by ultraviolet (<400 nm) and emits blue light (<500 nm), whereasthe acceptor is efficiently excited by blue but not by ultraviolet lightand emits green light (>500 nm), for example, P4-3 and S65C.

In the tandem constructs of the invention, the donor and acceptormoieties are connected through a linker moiety. The linker moiety is,preferably, a peptide moiety, but can be another organic molecularmoiety, as well. In a preferred embodiment, the linker moiety includes acleavage recognition site specific for an enzyme or other cleavage agentof interest. A cleavage site in the linker moiety is useful because whena tandem construct is mixed with the cleavage agent, the linker is asubstrate for cleavage by the cleavage agent. Rupture of the linkermoiety results in separation of the fluorescent protein moieties that ismeasurable as a change in FRET.

When the cleavage agent of interest is a protease, the linker cancomprise a peptide containing a cleavage recognition sequence for theprotease. A cleavage recognition sequence for a protease is a specificamino acid sequence recognized by the protease during proteolyticcleavage. In particular, the linker can contain any of the amino acidsequences in TABLE III. The sites are recognized by the enzymes asindicated and the site of cleavage is marked by a hyphen. Other proteasecleavage sites also are known in the art and can be included in thelinker moiety.

                  TABLE III                                                       ______________________________________                                        Protease      Sequence                                                        ______________________________________                                        HIV-1 protease                                                                              SQNY-PIVQ (SEQ ID NO: 3)                                           KARVL-AEAMS (SEQ ID NO: 4)                                                   Prohormone convertase PSPREGKR-SY (SEQ ID NO: 5)                              Interleukin-1b-converting YVAD-G (SEQ ID NO: 6)                               enzyme                                                                        Adenovirus endopeptidase MFGG-AKKR (SEQ ID NO: 7)                             Cytomegalovirus assemblin GVVNA-SSRLA (SEQ ID NO: 8)                          Leishmanolysin LIAY-LKKAT (SEQ ID NO: 9)                                      b-Secretase for amyloid VKM-DAEF (SEQ ID NO: 10)                              precursor protein                                                             Thrombin FLAEGGGVR-GPRVVERH                                                    (SEQ ID NO: 11)                                                              Renin and DRVYIHPF-HL-VIH                                                     angiotensin-converting (SEQ ID NO: 12)                                        enzyme                                                                        Cathepsin D KPALF-FRL (SEQ ID NO: 13)                                         Kininogenases including QPLGQTSLMK-RPPGFSPFR- SVQVMKT                         kallikrein QEGS (SEQ ID NO: 14)                                             ______________________________________                                         See, e.g., Matayoshi et al. (1990) Science 247:954, Dunn et al. (1994)        Meth. Enzymol. 241:254, Seidah & Chretien (1994) Meth. Enzymol. 244:175,      Thornberry (1994) Meth. Enzymol. 244:615, Weber & Tihanyi (1994) Meth.        Enzymol. 244:595, Smith et al. (1994) Meth. Enzymol. 244:412, Bouvier et      al. (1995) Meth. Enzymol. 248:614, Hardy et al. (1994) in Amyloid Protein     Precursor in Development, Aging, and Alzheimer's Disease, ed. C. L.           Masters et al. pp. 190-198.                                              

In the case of a known protease with cleavage activity of unknown orpartially defined specificity, a library of randomized linker sequencescan be used in place of a predetermined linker sequence in the tandemfluorescent protein construct in order to determine the sequencescleaved by a protease. The method can be used with a recombinantprotease constructed with a novel cleavage specificity. This method canalso be used to determine the specificity of cleavage of an orphanprotein that reveals sequence homology to a known protease structure orgroup of proteases.

In one embodiment, a genetically engineered library of tandemfluorescent protein constructs having randomized linkers can be used todefine the function of an orphan protease. Optionally, the orphanprotease, especially to if is thought to be expressed relativelyinactive precursor, can be coexpressed with the tandem fluorescentprotein construct. The protease may also be coexpressed with the tandemconstruct and under the control of an inducable promoter.

As used herein, a "library" refers to a collection containing at least 5different members, preferably at least 100 different members and morepreferably at least 200 different members. Each member of a tandemfluorescent substrate library comprises 2 tandemly linked fluorescentprotein moieties separated by a peptide linker moiety of variable aminoacid composition. The amino acid sequences for the peptide linker moietymay be completely random or biased towards a particular sequence basedon the homology between other proteases and the protease being tested.The library can be chemically synthesized, which is particularlydesirable if d-amino acids are to be included. In most instances,however, the library will be expressed in bacteria or a mammalian cell.

For example, the library can contain linkers with a diverse collectionof amino acids in which most or all of the amino acid positions arerandomized. Alternatively, the library can contain variable peptidemoieties in which only a few, e.g., one to ten, amino acid positions arevaried, but in which the probability of substitution is very high.

Preferably, libraries of tandem fluorescent protein candidate substratesare created by expressing protein from libraries of recombinant nucleicacid molecules having expression control sequences operatively linked tonucleic acid sequences that code for the expression of differentfluorescent protein candidate substrates. Methods of making nucleic acidmolecules encoding a diverse collection of peptides are described in,for example, U.S. Pat. No. 5,432,018 (Dower et al.), U.S. Pat. No.5,223,409 (Ladner et al.) and International patent publication WO92/06176 (Huse et al.).

For expression of tandem fluorescent protein candidate substrates,recombinant nucleic acid molecules are used to transfect cells, suchthat a cell contains a member of the library. This produces, in turn, alibrary of host cells capable of expressing a library of differentfluorescent protein candidate substrates. The library of host cells isuseful in the screening methods of this invention.

In one method of creating such a library, a diverse collection ofoligonucleotides having random codon sequences are combined to createpolynucleotides encoding peptides having a desired number of amino acidsfor the lnker moiety. The oligonucleotides preferably are prepared bychemical syntheses. The polynucleotides encoding peptide linker moietyof variable composition can then be ligated to the 5' or 3' end of anucleic acid encoding one of the tandem fluorescent protein moieties,using methods known in the art. This creates a recombinant nucleic acidmolecule coding for the expression of a fluorescent protein candidatesubstrate having a variable linker peptide moiety fused to the amino orcarboxy- terminus of one of the tandem fluorescent proteins. Thisrecombinant nucleic acid molecule is then inserted into an expressionvector in which the second fluorescent has already been inserted tocreate a recombinant nucleic acid molecule comprising expression controlsequences operatively linked to the sequences encoding the tandemlyrepeated fluorescent proteins separated by the linker moieties (FIG.10).

To generate the collection of oligonucleotides which forms a series ofcodons encoding a random collection of amino acids that is ultimatelycloned into the vector, a codon motif is used, such as (NNK)_(x), whereN may be A, C, G, or T (nominally equimolar), K is G or T (nominallyequimolar), and x is the desired number of amino acids in the peptidemoiety, e.g., 15 to produce a library of 15-mer peptides. The thirdposition may also be G or C, designated "S". Thus, NNK or NNS (i) codefor all the amino acids, (ii) code for only one stop codon, and (iii)reduce the range of codon bias from 6:1 to 3:1. The expression ofpeptides from randomly generated mixtures of oligonucleotides inappropriate recombinant vectors is discussed in Oliphant et al., Gene44:177-183 (1986), incorporated herein by reference.

An exemplified codon motif (NNK)₆ produces 32 codons, one for each of 12amino acids, two for each of five amino acids, three for each of threeamino acids and one (amber) stop codon. Although this motif produces acodon distribution as equitable as available with standard methods ofoligonucleotide synthesis, it results in a bias for amino acids encodedby two or threes alternative codons.

An alternative approach to minimize the bias against one-codon residuesinvolves the synthesis of 20 activated tri-nucleotides, eachrepresenting the codon for one of the 20 genetically encoded aminoacids. These are synthesized by conventional means, removed from thesupport but maintaining the base and 5-HO-protecting groups, andactivating by the addition of 3'O-phosphoramidite (and phosphateprotection with beta-cyanoethyl groups) by the method used for theactivation of mononucleosides, as generally described in McBride andCaruthers, Tetrahedron Letters 22:245 (1983). Degenerate "oligocodons"are prepared using these trimers as building blocks. The trimers aremixed at the desired molar ratios and installed in the synthesizer. Theratios will usually be approximately equimolar, but may be a controlledunequal ratio to obtain the over- to under-representation of certainamino acids coded for by the degenerate oligonucleotide collection. Thecondensation of the trimers to form the oligocodons is done essentiallyas described for conventional synthesis employing activatedmononucleosides as building blocks. See generally, Atkinson and Smith,Oligonucleotide Synthesis, M. J. Gait, ed. p35-82 (1984). Thus, thisprocedure generates a population of oligonucleotides for cloning that iscapable of encoding an equal distribution (or a controlled unequaldistribution) of the possible peptide sequences.

Because protease cleavage recognition sequences generally are only a fewamino acids in length, the linker moiety can include the recognitionsequence within flexible spacer amino acid sequences, such as GGGGS (SEQID NO:15). For example, a linker moiety including a cleavage recognitionsequence for Adenovirus endopeptidase could have the sequence GGGGGGSMFGGAKKRSGGGG GG (SEQ ID NO:16).

Alternatively, the linker moiety can be an organic molecular moiety thatcan contain a cleavage site for an enzyme that is not a protease. Themolecular structure is selected so that the distance between thefluorescent moieties allows FRET (i.e., less than about 10 nm). Forexample, the linker moiety can contain a structure that is recognized byb-lactamase, rendering the tandem complex a substrate for this enzyme.One structure for such a linker moiety is: ##STR1## in which one of Xand Y is the donor moiety and the other is the acceptor moiety. R' canbe, for example, H, lower alkyl or lower alkoxy of up to 15 carbon. R"can be H, physiologically-acceptable metal and ammonium cations, alkyl,alkoxy or aromatic groups of up to 15 carbon atoms. (See, e.g.,Bundgaard, H., Design of prodrugs, Elsevier Science publishers (1985);Bioreversible Carriers in Drug Design, New York:Pergamon Press (1987);Ferres, H. (1980) Chem. Ind. June:435-440.) Z' and Z" are parts of thelinker moiety having fewer than about 20 carbon atoms. Z" includes aheteroatom, such as oxygen or, preferably, sulfur, attached to thecephalosporin side chain to act as a nucleofuge. Such linker moietiesalso are described in U.S. patent application Ser. No. 08/407,547, filedMar. 20, 1995.

This invention contemplates tandem fluorescent protein constructsproduced in the form of a fusion protein by recombinant DNA technologyas well as constructs produced by chemically coupling fluorescentproteins to a linker. In either case, the fluorescent proteins for useas donor or acceptor moieties in a tandem construct of the inventionpreferably are produced recombinantly.

Recombinant production of fluorescent proteins involves expressingnucleic acids having sequences that encode the proteins. Nucleic acidsencoding fluorescent proteins can be obtained by methods known in theart. For example, a nucleic acid encoding the protein can be isolated bypolymerase chain reaction of cDNA from A. victoria using primers basedon the DNA sequence of A. victoria green fluorescent protein, aspresented in FIG. 1. PCR methods are described in, for example, U.S.Pat. No. 4,683,195; Mullis et al. (1987) Cold Spring Harbor Symp. Quant.Biol. 51:263; and Erlich, ed., PCR Technology, (Stockton Press, NewYork, 1989). Mutant versions of fluorescent proteins can be made bysite-specific mutagenesis of other nucleic acids encoding fluorescentproteins, or by random mutagenesis caused by increasing the error rateof PCR of the original polynucleotide with 0.1 mM MnCl₂ and unbalancednucleotide concentrations. See, e.g., U.S. patent application Ser. No.08/337,915, filed Nov. 10, 1994 or International applicationPCT/US95/14692, filed Nov. 10, 1995.

The construction of expression vectors and the expression of genes intransfected cells involves the use of molecular cloning techniques alsowell known in the art. Sambrook et al., Molecular Cloning--A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,(Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (most recent Supplement).

Nucleic acids used to transfect cells with sequences coding forexpression of the polypeptide of interest generally will be in the formof an expression vector including expression control sequencesoperatively linked to a nucleotide sequence coding for expression of thepolypeptide. As used, the term "nucleotide sequence coding forexpression of" a polypeptide refers to a sequence that, upontranscription and translation of mRNA, produces the polypeptide. Thiscan include sequences containing, e.g., introns. As used herein, theterm "expression control sequences" refers to nucleic acid sequencesthat regulate the expression of a nucleic acid sequence to which it isoperatively linked. Expression control sequences are "operativelylinked" to a nucleic acid sequence when the expression control sequencescontrol and regulate the transcription and, as appropriate, translationof the nucleic acid sequence. Thus, expression control sequences caninclude appropriate promoters, enhancers, transcription terminators, astart codon (i.e., ATG) in front of a protein-encoding gene, splicingsignals for introns, maintenance of the correct reading frame of thatgene to permit proper translation of the mRNA, and stop codons.

Recombinant fluorescent protein can be produced by expression of nucleicacid encoding the protein in E. coli. The fluorophore ofAequorea-related fluorescent proteins results from cyclization andoxidation of residues 65-67. Aequorea-related fluorescent proteins arebest expressed by cells cultured between about 20° C. and 30° C. Aftersynthesis, these enzymes are stable at higher temperatures (e.g., 37°C.) and can be used in assays at those temperatures.

The construct can also contain a tag to simplify isolation of the tandemconstruct. For example, a polyhistidine tag of, e.g., six histidineresidues, can be incorporated at the amino terminal end of thefluorescent protein. The polyhistidine tag allows convenient isolationof the protein in a single step by nickel-chelate chromatography.

A. Recombinant Nucleic Acids Encoding Tandem Construct Fusion Proteins

In a preferred embodiment, the tandem construct is a fusion proteinproduced by recombinant DNA technology in which a single polypeptideincludes a donor moiety, a peptide linker moiety and an acceptor moiety.The donor moiety can be positioned at the amino-terminus relative to theacceptor moiety in the polypeptide. Such a fusion protein has thegeneralized structure: (amino terminus) donor fluorescent proteinmoiety--peptide linker moiety--acceptor fluorescent protein moiety(carboxy terminus). Alternatively, the donor moiety can be positioned atthe carboxy-terminus relative to the acceptor moiety within the fusionprotein. Such a fusion protein has the generalized structure: (aminoterminus) acceptor fluorescent protein moiety--peptide linkermoiety--donor fluorescent protein moiety (carboxy terminus). Theinvention also envisions fusion proteins that contain extra amino acidsequences at the amino and/or carboxy termini, for example,polyhistidine tags.

Thus, tandem constructs encoded by a recombinant nucleic acid includesequences coding for expression of a donor fluorescent protein moiety,an acceptor fluorescent protein moiety and a peptide linker moiety. Theelements are selected so that upon expression into a fusion protein, thedonor and acceptor moieties exhibit FRET when the donor moiety isexcited.

The recombinant nucleic acid can be incorporated into an expressionvector comprising expression control sequences operatively linked to therecombinant nucleic acid. The expression vector can be adapted forfunction in prokaryotes or eukaryotes by inclusion of appropriatepromoters, replication sequences, markers, etc.

The expression vector can be transfected into a host cell for expressionof the recombinant nucleic acid. Host cells can be selected for highlevels of expression in order to purify the tandem construct fusionprotein. E. coli is useful for this purpose. Alternatively, the hostcell can be a prokaryotic or eukaryotic cell selected to study theactivity of an enzyme produced by the cell. In this case, the linkerpeptide is selected to include an amino acid sequence recognized by theprotease. The cell can be, e.g., a cultured cell or a cell in vivo.

A primary advantage of tandem construct fusion proteins is that they areprepared by normal protein biosynthesis, thus completely avoidingorganic synthesis and the requirement for customized unnatural aminoacid analogs. The constructs can be expressed in E. coli in large scalefor in vitro assays. Purification from bacteria is simplified when thesequences include polyhistidine tags for one-step purification bynickel-chelate chromatography. Alternatively, the substrates can beexpressed directly in a desired host cell for assays in situ, which isparticularly advantageous if the proteases of interest aremembrane-bound or regulated in a complex fashion or not yet abundant aspurified stable enzymes. No other generalizable method for continuousnondestructive assay of protease activity in living cells or organismspresently exists.

B. Non-Recombinant Coupling Methods

Fluorescent proteins can be attached through non-recombinant means. Inone embodiment, the moieties are attached to a linker by chemical means.This is preferred if the linker moiety is not a peptide. In this case,the linker moiety can comprise a cross-linker moiety. A number ofcross-linkers are well known in the art, including homo- orhetero-bifunctional cross-linkers, such as BMH, SPDP, etc. In general,the linker should have a length so as to separate the moieties by about10 Å to about 100 Å. This is more critical than the particular chemicalcomposition of the linker. Chemical methods for specifically linkingmolecules to the amino- or carboxy-terminus of a protein are reviewed byR. E. Offord, "Chemical Approaches to Protein Engineering," in ProteinEngineering--A Practical Approach, (1992) A. R. Rees, M. Sternberg andR. Wetzel, eds., Oxford University Press.

When the protein moieties are to be chemically coupled, fluorescentproteins can be isolated from natural sources by means known in the art.One method involves purifying the proteins to electrophoretichomogeneity. Also, J. R. Deschamps et al. describe a method of purifyingrecombinant Aequorea GFP in Protein Expression and Purification, (1995)6:555-558.

In another embodiment, the moieties are coupled by attaching each to anucleic acid molecule. The nucleic acids have sequences of sufficientlength and areas of sufficient complementarity to allow hybridizationbetween them, thereby linking the moieties through hydrogen bonds. Whenthe linker contains the sequence of a restriction site, this embodimentallows one to assay for the presence of restriction enzymes bymonitoring FRET after the nucleic acid is cleaved and the moietiesphysically separate.

In another embodiment, the moieties are coupled by attaching each to apolypeptide pair capable of bonding through dimerization. For example,the peptide can include sequences that form a leucine zipper, shown toenable dimerization of a protein to which it was attached. See A.Blondel et al., "Engineering the quaternary structure of an exportedprotein with a leucine zipper," Protein Engineering (1991) 4:457-461.The linker containing the leucine zipper in the Blondel et al. articlehad the sequence: IQRMKQLED KVEELLSKNY HLENEVARLK KLVGER (SEQ ID NO:17).In another embodiment, a peptide linker moiety can comprise the sequenceSKVILF (SEQ OID NO:18), which also is capable of dimerization. See WO94/28173.

C. Alternative Fluorescent Protein Constructs

This invention also contemplates tandem constructs possessing a singlefluorescent protein moiety that functions as donor or acceptor and anon-protein compound fluorescent moiety that functions as donor orquencher. In one embodiment, the construct comprises a donor fluorescentprotein moiety, a non-protein compound acceptor fluorescent moiety and alinker moiety that couples the donor and acceptor moieties.Alternatively, a tandem construct can comprise a non-protein compounddonor fluorescent moiety, an acceptor fluorescent protein moiety and alinker moiety that couples the donor and acceptor moieties. Non-proteincompound fluorescent donor moieties of particular interest includecoumarins and fluoresceins; particular quenchers of interest includefluoresceins, rhodols, rhodamines and azo dyes. Acceptable fluorescentdyes are described, for example, in U.S. application Ser. No.08/407,544, filed Mar. 20, 1995 (allowed, but yet to issue). The donorand acceptor moieties of these constructs are chosen with many of thesame considerations for FRET as for tandem fluorescent proteinconstructs having two fluorescent protein moieties.

III. ENZYMATIC ASSAYS USING TANDEM FLUORESCENT PROTEIN CONSTRUCTS

Tandem fluorescent protein constructs are useful in enzymatic assays.These assays take advantage of the fact that cleavage of the linkermoiety and separation of the fluorescent moieties results in ameasurable change in FRET. Methods for determining whether a sample hasactivity of an enzyme involve contacting the sample with a tandemfluorescent protein construct in which the linker moiety that couplesthe donor and acceptor moieties contains a cleavage recognition sitespecific for the enzyme. Then the donor moiety is excited with light inits excitation spectrum. If the linker moiety is cleaved, the donor andacceptor are free to drift apart, increasing the distance between thedonor and acceptor and preventing FRET. Then, the degree of FRET in thesample is determined. A degree of FRET that is lower than the amountexpected in a sample in which the tandem construct is not cleavedindicates that the enzyme is present.

The amount of activity of an enzyme in a sample can be determined bydetermining the degree of FRET in the sample at a first and second timeafter contact between the sample and the tandem construct, determiningthe difference in the degree of FRET. The amount of enzyme in the samplecan be calculated as a function of the difference in the degree of FRETusing appropriate standards. The faster or larger the loss of FRET, themore enzyme activity must have been present in the sample.

The degree of FRET can be determined by any spectral or fluorescencelifetime characteristic of the excited construct, for example, bydetermining the intensity of the fluorescent signal from the donor, theintensity of fluorescent signal from the acceptor, the ratio of thefluorescence amplitudes near the acceptor's emission maxima to thefluorescence amplitudes near the donor's emission maximum, or theexcited state lifetime of the donor.

For example, cleavage of the linker increases the intensity offluorescence from the donor, decreases the intensity of fluorescencefrom the acceptor, decreases the ratio of fluorescence amplitudes fromthe acceptor to that from the donor, and increases the excited statelifetime of the donor.

Preferably, changes in the degree of FRET are determined as a functionof the change in the ratio of the amount of fluorescence from the donorand acceptor moieties, a process referred to as "ratioing." Changes inthe absolute amount of substrate, excitation intensity, and turbidity orother background absorbances in the sample at the excitation wavelengthaffect the intensities of fluorescence from both the donor and acceptorapproximately in parallel. Therefore the ratio of the two emissionintensities is a more robust and preferred measure of cleavage thaneither intensity alone.

The excitation state lifetime of the donor moiety is, likewise,independent of the absolute amount of substrate, excitation intensity,or turbidity or other background absorbances. Its measurement requiresequipment with nanosecond time resolution.

Fluorescence in a sample is measured using a fluorimeter. In general,excitation radiation, from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscause the excitation radiation to excite the sample. In response,fluorescent proteins in the sample emit radiation which has a wavelengththat is different from the excitation wavelength. Collection optics thencollect the emission from the sample. The device can include atemperature controller to maintain the sample at a specific temperaturewhile it is being scanned. According to one embodiment, a multi-axistranslation stage moves a microtiter plate holding a plurality ofsamples in order to position different wells to be exposed. Themulti-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer. The computeralso can transform the data collected during the assay into anotherformat for presentation.

Methods of performing assays on fluorescent materials are well known inthe art and are described in, e.g., Lakowicz, J. R., Principles ofFluorescence Spectroscopy, New York:Plenum Press (1983); Herman, B.,Resonance energy transfer microscopy, in: Fluorescence Microscopy ofLiving Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed.Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp.219-243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park:Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.

Enzymatic assays also can be performed on living cells in vivo, or fromsamples derived from organisms transfected to express the tandemconstruct. Because tandem construct fusion proteins can be expressedrecombinantly inside a cell, the amount of enzyme activity in the cellor organism of which it is a part can be determined by determiningchanges in fluorescence of cells or samples from the organism.

In one embodiment, a cell is transiently or stably transfected with anexpression vector encoding a tandem fluorescent protein constructcontaining a linker moiety that is specifically cleaved by the enzyme tobe assayed. This expression vector optionally includes controllingnucleotide sequences such as promotor or enhancing elements. The enzymeto be assayed may either be intrinsic to the cell or may be introducedby stable transfection or transient co-transfection with anotherexpression vector encoding the enzyme and optionally includingcontrolling nucleotide sequences such as promoter or enhancer elements.The fluorescent protein construct and the enzyme preferably areexpressed in the same cellular compartment so that they have moreopportunity to come into contact.

If the cell does not possess enzyme activity, the efficiency of FRET inthe cell is high, and the fluorescence characteristics of the cellreflect this efficiency. If the cell possesses a high degree of enzymeactivity, most of the tandem construct expressed by the cell will becleaved. In this case, the efficiency of FRET is low, reflecting a largeamount or high efficiency of the cleavage enzyme relative to the rate ofsynthesis of the tandem fluorescent protein construct. If the level ofenzyme activity in the cell is such that an equilibrium is reachedbetween expression and cleavage of the tandem construct, thefluorescence characteristics will reflect this equilibrium level. In oneaspect, this method can be used to compare mutant cells to identifywhich ones possess greater or less enzymatic activity. Such cells can besorted by a fluorescent cell sorter based on fluorescence.

A contemplated variation of the above assay is to use the controllingnucleotide sequences to produce a sudden increase in the expression ofeither the tandem fluorescent protein construct or the enzyme beingassayed, e.g., by inducing expression of the construct. The efficiencyof FRET is monitored at one or more time intervals after the onset ofincreased expression. A low efficiency or rapid decline of FRET reflectsa large amount or high efficiency of the cleavage enzyme. This kineticdetermination has the advantage of minimizing any dependency of theassay on the rates of degradation or loss of the fluorescent proteinmoieties.

Libraries of host cells expressing tandem fluorescent protein candidatesubstrates are useful in identifying linker sequences that can becleaved by a target protease. In general, one begins with a library ofrecombinant host cells, each of which expresses a different fluorescentprotein candidate substrate. Each cell is expanded into a clonalpopulation that is genetically homogeneous. The method consists ofmeasuring FRET from each clonal population before and at least onespecified time after a known change in intracellular protease activity.This could be achieved using a fluorimeter, a 96 well plate reader, orby FACS (fluorescence Activated Cell Sorting) anlysis and sorting. Thischange in protease activity could be produced by transfection with agene encoding the protease, or infection of a cell by a virus, orinduction of protease gene expression using expression control elements,or by any condition that post-translationally modulates the activity ofa protease that has already been expressed. An example of the latter isthe activation of Calpain 1 by increases in intracellular calcium. Thenucleic acids from cells exhibiting a change in FRET can be isolated forexample by PCR amplification, and the linker sequences that could becleaved by the protease identified by sequencing. The results from thesestudies could used as the basis for the generation of more targetedlibraries to identify optimal cleavage motifs through repeated rounds ofanalysis and selection of clones exhibiting the largest and most rapidchanges in FRET in the presence, but not the absence of the protease.

In another embodiment, the vector may be incorporated into an entireorganism by standard transgenic or gene replacement techniques. Anexpression vector capable of expressing the enzyme optionally may beincorporated into the entire organism by standard transgenic or genereplacement techniques. Then, a sample from the organism containing thetandem construct or the cleaved moieties is tested. For example, cell ortissue homogenates, individual cells, or samples of body fluids, such asblood, can be tested.

The enzymatic assays of the invention can be used in drug screeningassays to identify compounds that alter the activity of an enzyme. Inone embodiment, the assay is performed on a sample in vitro containingthe enzyme. A sample containing a known amount of enzyme is mixed with atandem construct of the invention and with a test compound. The amountof the enzyme activity in the sample is then determined as above, e.g.,by determining the degree of fluorescence at a first and second timeafter contact between the sample, the tandem construct and the compound.Then the amount of activity per mole of enzyme in the presence of thetest compound is compared with the activity per mole of enzyme in theabsence of the test compound. A difference indicates that the testcompound alters the activity of the enzyme.

In another embodiment, the ability of a compound to alter enzymeactivity in vivo is determined. In an in vivo assay, cells transfectedwith a expression vector encoding a tandem construct of the inventionare exposed to different amounts of the test compound, and the effect onfluorescence in each cell can be determined. Typically, the differenceis calibrated against standard measurements to yield an absolute amountof enzyme activity. A test compound that inhibits or blocks theexpression of the enzyme can be detected by increased FRET in treatedcells compared to untreated controls.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLES Example 1 Construction of Tandem Fluorescent Protein Constructs

Mutant Green Fluorescent Proteins were created as follows. Randommutagenesis of the Aequorea green fluorescent protein (FIG. 1) wasperformed by increasing the error rate of the PCR with 0.1 mM MnCl₂ andunbalanced nucleotide concentrations. The templates used for PCR encodedthe GFP mutants S65T, Y66H and Y66W. They had been cloned into the BamH1site of the expression vector PRSETB (Invitrogen), which includes a T7promoter and a polyhistidine tag. The GFP coding region (shown in bold)was flanked by the following 5' and 3' sequences: 5'-G GAT CCC CCC GCTGAA TTC ATG (SEQ ID NO:19) . . . AAA TAA TAA GGA TCC (SEQ ID NO:20) -3'.The 5' primer for the mutagenic PCR was the T7 primer matching thevector sequence; the 3' primer was 5'-GGT AAG CTT TTA TTT GTA TAG TTCATC CAT GCC-3' (SEQ ID NO:21), specific for the 3' end of GFP, creatinga HindIII restriction site next to the stop codon.

Amplification was over 25 cycles (1 min at 94° C., 1 min 52° C., 1 min72° C.) using the AmpliTaq polymerase from Perkin Elmer). Four separatereactions were run in which the concentration of a different nucleotidewas lowered from 200 μM to 50 μM. The PCR products were combined,digested with BamHI and HindIII and ligated to the pRSETB cut with BamHIand HindIII. The ligation mixture was dialyzed against water, dried andsubsequently transformed into the bacterial strain BL21(DE3) byelectroporation (50 μl electrocompetent cells in 0.1 cm cuvettes, 1900V, 200 ohm, 25 μF). Colonies on agar were visually screened forbrightness as previously described. R. Heim et al., "Wavelengthmutations and post-translational autooxidation of green fluorescentprotein," Proc Natl Acad Sci USA 1994, 91:12501-12504. On the order of7000 colonies were examined in each successful round of mutagenesis,which is not claimed to be exhaustive. The selected clones weresequenced with the Sequenase version 2.0 kit from U.S. Biochemical.

A nucleic acid sequence encoding a tandem GFP-BFP construct fusionprotein was produced as follows. The DNA of the GFP mutant S65C (Heim R,Cubitt A B, Tsien R Y, "Improved green fluorescence," Nature 1995,373:663-664) was amplified by PCR (1 cycle 3 min 94° C., 2 min 33° C., 2min 72° C.; 20 cycles 1 min 94° C., 1 min 44° C., 1 min 72° C.) with Pfupolymerase (Stratagene) using the primers 5'-AGA AAG GCT AGC AAA GGA GAAGAA C-3' (SEQ ID NO:22) and 5'-T CAG TCT AGA TTT GTA TAG TTC ATC-3' (SEQID NO:23) to create a NheI site and a (NheI compatible) XbaI site and toeliminate the GFP stop codon. The restricted product was cloned in-frameinto the NheI site of the construct pRSETB-Y66H/Y145F, between apolyhistidine tag and an enterokinase cleavage site. When translatedthis fusion gives the following sequence: MRGSHHHHHH GMA (SEQ IDNO:24)--(S2 . . . GFP:S65C . . . K238 "S65C")--SSMTGGQQMG RDLYDDDDKDPPAEF (SEQ ID NO:25)--(GFP:Y66H/Y145F "P4-3") The linker moiety includescleavage recognition sites for many proteases, including trypsin,enterokinase and calpain: ##STR2## Several other constructs wereconstructed and tested using the same linker moiety. One of these hasthe structure S65C--linker--P4. Another had the structureS65C--linker--W7. A third construct had the structure S65T--linker--W7.A fourth construct had the structure P4-3--linker--W7.

Cultures with freshly transformed E. coli cells were grown at 37° C. toan optical density of 0.8 at 600 nm, then induced with 0.4 mMisopropylthiogalactoside overnight at room temperature. Expressionlevels were roughly equivalent between mutants and are typical for theT7 expression system used. Cells were washed in PBS pH 7.4, resuspendedin 50 mM Tris pH 8.0, 300 mM NaCl and lysed in a French press. Thepolyhistidine-tagged GFP proteins were purified from cleared lysates onnickel-chelate columns (Qiagen) using 100 mM imidazole in the abovebuffer to elute the protein. Samples used for proteolytic experimentswere further purified by MonoQ FPLC to remove monomeric GFP. Proteinconcentrations were estimated with bicinchoninic acid (BCA kit fromPierce) using bovine serum albumin as a standard.

Example 2 Cleavage Measurements

Proteolytic cleavage of 10 μg of the various GFP-BFP fusion proteinswere performed in 500 μl PBS pH 7.4 with 0.1 μg trypsin (Sigma, gradeIII) and emission spectra were recorded at different time intervals.Analogous cleavage experiments were done also with enterokinase (Sigma)and calpain.

Excitation spectra were obtained by collecting emission at therespective peak wavelengths and were corrected by a Rhodamine B quantumcounter. Emission spectra were likewise measured at the respectiveexcitation peaks and were corrected using factors from the fluorometermanufacturer (Spex Industries, Edison, N.J.). In cleavage experimentsemission spectra were recorded at excitation 368 nm or at 432 nm. Formeasuring molar extinction coefficients, 20 to 30 μg of protein wereused in 1 ml of PBS pH 7.4. The extinction coefficients in TABLE Inecessarily assume that the protein is homogeneous and properly folded;if this assumption is incorrect, the real extinction coefficients couldbe yet higher. Quantum yields of wild-type GFP, S65T, and P4-1 mutantswere estimated by comparison with fluorescein in 0.1 N NaOH as astandard of quantum yield 0.91. J. N. Miller, ed., Standards inFluorescence Spectrometry, New York: Chapman and Hall (1981). Mutants P4and P4-3 were likewise compared to 9-aminoacridine in water (quantumyield 0.98). W2 and W7 were compared to both standards, which gaveconcordant results.

Excited at 368 nm, the uncleaved S65C--linker--P4-3 construct emittedbright green light that gradually dimmed upon cleavage of the linker toseparate the protein domains. As the cleavage by trypsin progressed (0,2, 5, 10, and 47 min), more blue light was emitted. There was no furtherchange after 47 minutes.

The emission spectrum of the intact fusion protein (FIG. 3) shows thatFRET is fairly efficient, because UV excitation causes substantial greenemission from the acceptor S65C. After proteolytic cleavage of thespacer, which permits the two domains to diffuse apart, the greenemission almost completely disappears, whereas the blue emission fromthe Y66H/Y145F is enhanced because its excited state is no longer beingquenched by the acceptor. Control experiments with the same proteolyticconditions applied to either GFP mutant alone showed no effect, arguingthat the GFP domains per se are resistant to proteolysis, as is known tobe the case for the native protein. W. W. Ward et al., "Spectralperturbations of the Aequorea green-fluorescent protein," Photochem.Photobiol. (1982) 35:803-808.

Similar result were obtained when the S65C--linker--P4-3 fusionconstruct was cleaved with calpain and excited at 368 nm. (See FIG. 4.)

The tandem construct S65C--linker--P4 was exposed to enterokinase andexcited at 368 nm. FRET diminished over time, demonstrating that onecould detect cleavage of the linker by enterokinase. (See FIG. 5.)

The tandem construct S65T--linker--W7 was exposed to trypsin and excitedat 432 nm. Cleavage of the linker and separation of the moieties wasdetectable as a decrease in FRET over time. (See FIG. 6.)

The tandem construct P4-3--linker--W7 was exposed to trypsin and excitedat 368 nm. FIG. 7. demonstrates the change in FRET resulting fromcleavage.

The tandem construct W1B--linker--10c was exposed to trypsin and excitedat 433 nm. FIG. 8. demonstrates the change in FRET resulting fromcleavage.

FIG. 9 depicts fluorescent ratio changes upon cleavage of a compositioncontaining the tandem construct W1B--linker--10c fluorescent constructat different protein concentrations after exposure to trypsin measuredin a fluorescent 96 well microtitre plate reader (a CytoFluor II Series4000 Perseptive Biosystems. Microtitre wells were excited with light at395+/-25 nm, and the emitted light measured at 460+/-20 nm and 530+/-15nm using appriopriate excitation and emission filter sets.

These different tandem fluorescent protein constructs demonstrate thatfluorescence resonance energy transfer can monitor the distance betweenfluorescent protein domains. Disruption of FRET between man-madechromophores in a short synthetic peptide has been used before to assayproteases (G. A. Krafft et al., "Synthetic approaches to continuousassays of retroviral proteases," Methods Enzymol. (1994) 241:70-86; C.G. Knight, "Fluorimetric assays of proteolytic enzymes," MethodsEnzymol. (1995) 248:18-34), but use of fluorescent proteins as thefluorophores gives the unique possibility of replacing organic synthesisby molecular biology and monitoring proteases in situ in living cellsand organisms. FRET is also one of the few methods for imaging dynamicnon-covalent protein-protein associations in situ.

The present invention provides novel tandem fluorescent proteinconstructs and methods for their use. While specific examples have beenprovided, the above description is illustrative and not restrictive.Many variations of the invention will become apparent to those skilledin the art upon review of this specification. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but instead should be determined with reference to theappended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 716 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..717                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATG AGT AAA GGA GAA GAA CTT TTC ACT GGA GT - #T GTC CCA ATT CTT        GTT       48                                                                    Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Va - #l Val Pro Ile Leu Val            1               5 - #                 10 - #                 15              - - GAA TTA GAT GGT GAT GTT AAT GGG CAC AAA TT - #T TCT GTC AGT GGA GAG           96                                                                       Glu Leu Asp Gly Asp Val Asn Gly His Lys Ph - #e Ser Val Ser Gly Glu                        20     - #             25     - #             30                  - - GGT GAA GGT GAT GCA ACA TAC GGA AAA CTT AC - #C CTT AAA TTT ATT TGC          144                                                                       Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Th - #r Leu Lys Phe Ile Cys                    35         - #         40         - #         45                      - - ACT ACT GGA AAA CTA CCT GTT CCA TGG CCA AC - #A CTT GTC ACT ACT TTC          192                                                                       Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Th - #r Leu Val Thr Thr Phe                50             - #     55             - #     60                          - - TCT TAT GGT GTT CAA TGC TTT TCA AGA TAC CC - #A GAT CAT ATG AAA CGG          240                                                                       Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pr - #o Asp His Met Lys Arg            65                 - # 70                 - # 75                 - # 80       - - CAT GAC TTT TTC AAG AGT GCC ATG CCC GAA GG - #T TAT GTA CAG GAA AGA          288                                                                       His Asp Phe Phe Lys Ser Ala Met Pro Glu Gl - #y Tyr Val Gln Glu Arg                            85 - #                 90 - #                 95              - - ACT ATA TTT TTC AAA GAT GAC GGG AAC TAC AA - #G ACA CGT GCT GAA GTC          336                                                                       Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Ly - #s Thr Arg Ala Glu Val                       100      - #           105      - #           110                  - - AAG TTT GAA GGT GAT ACC CTT GTT AAT AGA AT - #C GAG TTA AAA GGT ATT          384                                                                       Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Il - #e Glu Leu Lys Gly Ile                   115          - #       120          - #       125                      - - GAT TTT AAA GAA GAT GGA AAC ATT CTT GGA CA - #C AAA TTG GAA TAC AAC          432                                                                       Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly Hi - #s Lys Leu Glu Tyr Asn               130              - #   135              - #   140                          - - TAT AAC TCA CAC AAT GTA TAC ATC ATG GCA GA - #C AAA CAA AAG AAT GGA          480                                                                       Tyr Asn Ser His Asn Val Tyr Ile Met Ala As - #p Lys Gln Lys Asn Gly           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - ATC AAA GTT AAC TTC AAA ATT AGA CAC AAC AT - #T GAA GAT GGA AGC        GTT      528                                                                    Ile Lys Val Asn Phe Lys Ile Arg His Asn Il - #e Glu Asp Gly Ser Val                          165  - #               170  - #               175              - - CAA CTA GCA GAC CAT TAT CAA CAA AAT ACT CC - #A ATT GGC GAT GGC CCT          576                                                                       Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pr - #o Ile Gly Asp Gly Pro                       180      - #           185      - #           190                  - - GTC CTT TTA CCA GAC AAC CAT TAC CTG TCC AC - #A CAA TCT GCC CTT TCG          624                                                                       Val Leu Leu Pro Asp Asn His Tyr Leu Ser Th - #r Gln Ser Ala Leu Ser                   195          - #       200          - #       205                      - - AAA GAT CCC AAC GAA AAG AGA GAC CAC ATG GT - #C CTT CTT GAG TTT GTA          672                                                                       Lys Asp Pro Asn Glu Lys Arg Asp His Met Va - #l Leu Leu Glu Phe Val               210              - #   215              - #   220                          - - ACA GCT GCT GGG ATT ACA CAT GGC ATG GAT GA - #A CTA TAC AAA TA               - #716                                                                    Thr Ala Ala Gly Ile Thr His Gly Met Asp Gl - #u Leu Tyr Lys                   225                 2 - #30                 2 - #35                            - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 238 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Va - #l Val Pro Ile Leu Val        1               5 - #                 10 - #                 15              - - Glu Leu Asp Gly Asp Val Asn Gly His Lys Ph - #e Ser Val Ser Gly Glu                   20     - #             25     - #             30                  - - Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Th - #r Leu Lys Phe Ile Cys               35         - #         40         - #         45                      - - Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Th - #r Leu Val Thr Thr Phe           50             - #     55             - #     60                          - - Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pr - #o Asp His Met Lys Arg       65                 - # 70                 - # 75                 - # 80       - - His Asp Phe Phe Lys Ser Ala Met Pro Glu Gl - #y Tyr Val Gln Glu Arg                       85 - #                 90 - #                 95              - - Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Ly - #s Thr Arg Ala Glu Val                  100      - #           105      - #           110                  - - Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Il - #e Glu Leu Lys Gly Ile              115          - #       120          - #       125                      - - Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly Hi - #s Lys Leu Glu Tyr Asn          130              - #   135              - #   140                          - - Tyr Asn Ser His Asn Val Tyr Ile Met Ala As - #p Lys Gln Lys Asn Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ile Lys Val Asn Phe Lys Ile Arg His Asn Il - #e Glu Asp Gly Ser        Val                                                                                             165  - #               170  - #               175             - - Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pr - #o Ile Gly Asp Gly Pro                  180      - #           185      - #           190                  - - Val Leu Leu Pro Asp Asn His Tyr Leu Ser Th - #r Gln Ser Ala Leu Ser              195          - #       200          - #       205                      - - Lys Asp Pro Asn Glu Lys Arg Asp His Met Va - #l Leu Leu Glu Phe Val          210              - #   215              - #   220                          - - Thr Ala Ala Gly Ile Thr His Gly Met Asp Gl - #u Leu Tyr Lys              225                 2 - #30                 2 - #35                            - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Ser Gln Asn Tyr Pro Ile Val Gly                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Lys Ala Arg Val Leu Ala Glu Ala Met Ser                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Pro Ser Pro Arg Glu Gly Lys Arg Ser Tyr                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Tyr Val Ala Asp Gly                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Met Phe Gly Gly Ala Lys Lys Arg                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Gly Val Val Asn Ala Ser Ser Arg Leu Ala                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Leu Ile Ala Tyr Leu Lys Lys Ala Thr                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Val Lys Met Asp Ala Glu Phe                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pr - #o Arg Val Val Glu Arg      Hi                                                                            1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Asp Arg Val Tyr Ile His Pro Phe His Leu Va - #l Ile His                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Lys Pro Ala Leu Phe Phe Arg Leu                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Gln Pro Leu Gly Gln Thr Ser Leu Met Lys Ar - #g Pro Pro Gly Phe Ser      1                  - # 5                 - #           10                      - - Pro Phe Arg Ser Val Gln Val Met Lys Thr Gl - #n Glu Gly Ser                          20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Gly Gly Gly Gly Ser                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Gly Gly Gly Gly Gly Gly Ser Met Phe Gly Gl - #y Ala Lys Lys Arg Ser      1               5   - #                10  - #                15               - - Gly Gly Gly Gly Gly Gly                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Ile Gln Arg Met Lys Gln Leu Glu Asp Lys Va - #l Glu Glu Leu Leu Ser      1               5   - #                10  - #                15               - - Lys Asn Tyr His Leu Glu Asn Glu Val Ala Ar - #g Leu Lys Lys Leu Val                  20      - #            25      - #            30                   - - Gly Glu Arg                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Ser Lys Val Ile Leu Phe                                                  1                  - # 5                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (oligonucleotide)                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - GGATCCCCCC GCTGAATTCA TG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (oligonucleotide)                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - AAATAATAAG GATCC              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (primer)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - GGTAAGCTTT TATTTGTATA GTTCATCCAT GCC       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (primer)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - AGAAAGGCTA GCAAAGGAGA AGAA          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (primer)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - TCAGTCTAGA TTTGTATAGT TCATC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Met Arg Gly Ser His His His His His His                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Ser Ser Met Thr Gly Gly Gln Gln Met Gly Ar - #g Asp Leu Tyr Asp Asp      1               5   - #                10  - #                15               - - Asp Asp Lys Asp Pro Pro Ala Glu Phe                                                  20      - #            25                                        __________________________________________________________________________

What is claimed is:
 1. A tandem fluorescent protein construct,comprising:a donor fluorescent protein moiety, an acceptor fluorescentprotein moiety, and a peptide linker moiety coupling said donorfluorescent protein moiety and said acceptor fluorescent protein moiety,wherein cyclized amino acids of said donor fluorescent protein moietyemit light characteristic of said donor fluorescent protein moiety,further wherein said donor fluorescent protein moiety and said acceptorfluorescent protein moiety exhibit fluorescence resonance energytransfer when said donor fluorescent protein moiety is excited, and saidlinker moiety does not substantially emit light to excite said donorfluorescent protein moiety.
 2. The tandem fluorescent protein constructof claim 1, wherein at least one of said donor fluorescent moiety andsaid acceptor fluorescent protein moiety comprises an Aequorea-relatedfluorescent protein moiety comprising a mutation at a position selectedfrom the group consisting of F64, S65, Y66, V68, S72, Y145, N146, N149,M153, V163, I167, T203, and N212.
 3. The tandem fluorescent proteinconstruct of claim 1, wherein both of said donor fluorescent proteinmoiety and said acceptor fluorescent protein moiety are anAequorea-related fluorescent protein moiety.
 4. The tandem fluorescentprotein construct of claim 3, wherein said mutation is selected from thegroup consisting of F64L, S65G, S65T, Y66F, Y66H, Y66W, V68L, S72A,Y145F, N146I, N149K, M153T, V163A, I167T, T203I, T203Y, and N212K. 5.The tandem fluorescent protein construct of claim 4, wherein saidmutation is selected for 10c, W1B, Emerald, and Sapphire.
 6. The tandemfluorescent protein construct of claim 5, wherein said donor fluorescentprotein moiety is P4-3, Sapphire, W7, or Y66H and said acceptor proteinfluorescent moiety is W7, Topaz, S65T, or S65C, with the proviso thatwhen said donor fluorescent protein moiety is P4-3, then said acceptorfluorescent protein moiety is not S65T or S65C and when said donorfluorescent protein moiety is W7, then said acceptor fluorescent moietyis not S65T.
 7. The tandem fluorescent protein construct of claim 1,wherein said linker moiety comprises a cleavage recognition site for anenzyme.
 8. The tandem fluorescent protein construct of claim 7, whereinsaid linker moiety is a peptide moiety.
 9. The tandem fluorescentprotein construct of claim 8, wherein said donor fluorescent proteinmoiety, said acceptor fluorescent protein moiety, and said linker moietycomprise a single polypeptide.
 10. The tandem fluorescent proteinconstruct of claim 9, wherein said linker moiety comprises between about5 and 50 amino acids.
 11. The tandem fluorescent protein construct ofclaim 10, wherein said linker moiety comprises between about 10 and 30amino acids.
 12. The tandem fluorescent protein construct of claim 9,wherein said linker moiety comprises a cleavage recognition site for anenzyme selected from the group consisting of trypsin, enterokinase,HIV-1 protease, prohormone convertase, interleukin-1b-converting enzyme,adenovirus endopeptidase, cytomegalovirus assemblin, leishmanolysin,β-secretase for amyloid precursor protein, thrombin, renin,angiotensin-converting enzyme, cathepsin D and a kininogenase.
 13. Thetandem fluorescent protein construct of claim 10, wherein said donorfluorescent protein moiety is positioned at the amino terminus of thepolypeptide relative to said acceptor fluorescent protein moiety. 14.The tandem fluorescent protein construct of claim 1, wherein saidpeptide linker moiety is of a length and orientation that allowsfluorescent energy transfer between said donor fluorescent proteinmoiety and said acceptor fluorescent protein moiety.
 15. The tandemfluorescent protein construct of claim 7, comprising a cleavagerecognition site for beta-lactamase.
 16. The tandem fluorescent proteinconstruct of claim 11, wherein said linker moiety comprises a proteaserecognition site.
 17. A recombinant nucleic acid encoding for theexpression of a functional tandem fluorescent protein construct, saidtandem fluorescent protein construct comprising:a donor fluorescentprotein moiety, an acceptor fluorescent protein moiety, and a peptidelinker moiety coupling said donor fluorescent protein moiety and saidacceptor fluorescent protein moiety, wherein said donor fluorescentprotein moiety and said acceptor fluorescent protein moiety exhibitfluorescence resonance energy transfer when said donor fluorescentprotein moiety is excited, and said peptide linker moiety does notsubstantially emit light to excite said donor fluorescent proteinmoiety, further wherein said peptide linker moiety comprises a cleavagerecognition site for a protease.
 18. The recombinant nucleic acid ofclaim 17, wherein at least one of said donor fluorescent moiety and saidacceptor fluorescent protein moiety comprises an Aequorea-relatedfluorescent protein moiety comprising a mutation at a position selectedfrom the group consisting of F64, S65, Y66, V68, S72, Y145, N146, N149,M153, V163, I167, T203, and N212.
 19. The recombinant nucleic acid ofclaim 7, wherein said peptide linker moiety is of a length andorientation that allows fluorescent resonance energy transfer betweensaid donor fluorescent protein moiety and said acceptor fluorescentprotein moiety.
 20. An expression vector, comprising: an expressioncontrol sequence operatively linked to a sequence coding for theexpression of a functional tandem fluorescent protein construct, saidtandem fluorescent protein construct comprising:a donor fluorescentprotein moiety, an acceptor fluorescent protein moiety, and a peptidelinker moiety coupling said donor fluorescent protein moiety and saidacceptor fluorescent protein moiety, wherein said donor fluorescentprotein moiety and said acceptor fluorescent protein moiety exhibitfluorescence resonance energy transfer when said donor fluorescentprotein moiety is excited, and said peptide linker moiety does notsubstantially emit light to excite said donor fluorescent protein moietyfurther wherein at least one of said donor fluorescent moiety and saidacceptor fluorescent protein moiety comprises an Aequorea-relatedfluorescent protein moiety comprising a mutation at a position selectedfrom the group consisting of F64, S65, Y66, V68, S72, Y145, N146, N149,M153, V163, I167, T203, and N212.
 21. A host cell transfected with anexpression vector, said expression vector comprising: an expressioncontrol sequence operatively linked to a sequence coding for theexpression of a functional tandem fluorescent protein construct, saidtandem fluorescent protein construct comprising:a donor fluorescentprotein moiety, an acceptor fluorescent protein moiety, and a peptidelinker moiety coupling said donor fluorescent protein moiety and saidacceptor fluorescent protein moiety, wherein said donor fluorescentprotein moiety and said acceptor fluorescent protein moiety exhibitfluorescence resonance energy transfer when said donor fluorescentprotein moiety is excited, and said peptide linker moiety does notsubstantially emit light to excite said donor fluorescent proteinmoiety, wherein said peptide linker moiety comprises a cleavagerecognition site for a protease.
 22. The host cell of claim 21, whereinat least one of said donor fluorescent moiety and said acceptorfluorescent protein moiety comprises an Aequorea-related fluorescentprotein moiety comprising a mutation at a position selected from thegroup consisting of F64, S65, Y66, V68, S72, Y145, N146, N149, M153,V163, I167, T203, and N212.
 23. A method for determining whether asample contains an enzyme, comprising:contacting a sample with a tandemfluorescent protein construct, said tandem fluorescent protein constructcomprising:a donor fluorescent protein moiety, an acceptor fluorescentprotein moiety, and a linker moiety comprising a cleavage recognitionsite for an enzyme, coupling said donor fluorescent protein moiety andsaid acceptor fluorescent protein moiety, wherein cyclized amino acidsof said donor fluorescent protein moiety emit light characteristic ofsaid donor fluorescent protein moiety, further wherein said donorfluorescent protein moiety and said acceptor fluorescent protein moietyexhibit fluorescence resonance energy transfer when said donorfluorescent protein moiety is excited, and said linker moiety does notsubstantially emit light to excite said donor fluorescent proteinmoiety, further wherein at least one of said donor fluorescent moietyand said acceptor fluorescent protein moiety comprises anAequorea-related fluorescent protein moiety comprising a mutation at aposition selected from the group consisting of F64, S65, Y66, V68, S72,Y145, N146, N149, M153, V163, I167, T203, and N212, exciting said donorfluorescent protein moiety, and determining a fluorescence property insaid sample, wherein the presence of said enzyme in said sample resultsin a change in the degree of fluorescence resonance energy transfer. 24.A method for determining the activity of an enzyme in a cell,comprising:providing a cell that expresses a tandem fluorescent proteinconstruct, said tandem fluorescent protein construct comprising:a donorfluorescent protein moiety, an acceptor fluorescent protein moiety, anda peptide linker moiety comprising a cleavage recognition amino acidsequence specific for said enzyme coupling said donor fluorescentprotein moiety and said acceptor fluorescent protein moiety, whereincyclized amino acids of said donor fluorescent protein moiety emit lightcharacteristic of said donor fluorescent protein moiety, further whereinsaid donor fluorescent protein moiety and said acceptor fluorescentprotein moiety exhibit fluorescence resonance energy transfer when saiddonor fluorescent protein moiety is excited, and said linker moiety doesnot substantially emit light to excite said donor fluorescent proteinmoiety, further wherein at least one of said donor fluorescent moietyand said acceptor fluorescent protein moiety comprises anAequorea-related fluorescent protein moiety comprising a mutation at aposition selected from the group consisting of F64, S65, Y66, V68, S72,Y145, N146, N149, M153, V163, I167, T203, and N212, exciting said donorfluorescent protein moiety, and determining the degree of fluorescenceresonance energy transfer in said cell, wherein the presence of saidactivity in said cell results in a change in the degree of fluorescenceresonance energy transfer.
 25. A method for determining the amount ofactivity of an enzyme in a sample from an organism,comprising:contacting a sample from an organism with a tandemfluorescent protein construct, said construct comprisinga donorfluorescent protein moiety, an acceptor fluorescent protein moiety, anda peptide linker moiety comprising a cleavage recognition amino acidsequence specific for said enzyme coupling said donor fluorescentprotein moiety and said acceptor fluorescent protein moiety, whereincyclized amino acids of said donor fluorescent protein moiety emit lightcharacteristic of said donor fluorescent protein moiety, further whereinsaid donor fluorescent protein moiety and said acceptor fluorescentprotein moiety exhibit fluorescence resonance energy transfer when saiddonor fluorescent protein moiety is excited, and said peptide linkermoiety does not substantially emit light to excite said donorfluorescent protein moiety, further wherein at least one of said donorfluorescent moiety and said acceptor fluorescent protein moietycomprises an Aequorea-related fluorescent protein moiety comprising amutation at a position selected from the group consisting of F64, S65,Y66, V68, S72, Y145, N146, N149, M153, V163, I167, T203, and N212, andexciting said donor fluorescent protein moiety, and determining thedegree of fluorescence resonance energy transfer in said sample, whereinthe presence of said activity in said sample results in a change in thedegree of fluorescence resonance energy transfer.
 26. A method fordetermining whether a compound alters the activity of an enzyme,comprising:contacting a sample containing an enzyme with a compound anda tandem fluorescent protein construct, said tandem fluorescent proteinconstruct comprising:a donor fluorescent protein moiety, an acceptorfluorescent protein moiety, and a linker moiety coupling said donorfluorescent protein moiety and said acceptor fluorescent protein moiety,wherein cyclized amino acids of said donor fluorescent protein moietyemit light characteristic of said donor fluorescent protein moiety,further wherein said donor fluorescent protein moiety and said acceptorfluorescent protein moiety exhibit fluorescence resonance energytransfer when said donor fluorescent protein moiety is excited, and saidlinker moiety does not substantially emit light to excite said donorfluorescent protein moiety, further wherein at least one of said donorfluorescent moiety and said acceptor fluorescent protein moietycomprises an Aequorea-related fluorescent protein moiety comprising amutation at a position selected from the group consisting of F64, S65,Y66, V68, S72, Y145, N146, N149, M153, V163, I167, T203, and N212, andexciting said donor fluorescent protein moiety, and determining afluorescent property of said sample,wherein an activity of said enzymeis determined by a change in the degree of said fluorescent property inthe presence and absence of said compound.
 27. A method for determiningwhether a compound alters the activity of an enzyme in a cell,comprising:providing a first and second cells that express a tandemfluorescent protein construct, said tandem fluorescent protein constructcomprising:a donor fluorescent protein moiety, an acceptor fluorescentprotein moiety, and a peptide linker moiety comprising a cleavagerecognition amino acid sequence specific for said enzyme coupling saiddonor fluorescent protein moiety and said acceptor fluorescent proteinmoiety, wherein cyclized amino acids of said donor fluorescent proteinmoiety emit light characteristic of said donor fluorescent proteinmoiety, further wherein said donor fluorescent protein moiety and saidacceptor fluorescent protein moiety exhibit fluorescence resonanceenergy transfer when said donor fluorescent protein moiety is excited,and said peptide linker moiety does not substantially emit light toexcite said donor fluorescent protein moiety, and exciting said donorfluorescent protein moiety, contacting said first cell with an amount ofsaid compound, contacting the second cell with a different amount ofsaid compound, further wherein at least one of said donor fluorescentmoiety and said acceptor fluorescent protein moiety comprises anAequorea-related fluorescent protein moiety comprising a mutation at aposition selected from the group consisting of F64, S65, Y66, V68, S72,Y145, N146, N149, M153, V163, I167, T203, and N212, and exciting saiddonor fluorescent protein moiety in said first and second cell,determining the degree of fluorescence resonance energy transfer in saidfirst and second cells, and comparing the degree of fluorescenceresonance energy transfer in said first cell and said second cell,wherein a difference in the degree of fluorescence resonance energytransfer in said first cell and said second cell indicates that thecompound alters the activity of said enzyme.